Global burden of tuberculosis

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An estimated 15 million active cases, leading to…..

An estimated 9 million new infections

Approx 2 million deaths

Approx 2 Billion USD in direct control costs

And an uncounted indirect cost in lost lives and productivity

Global burden of tuberculosis (The economist’s view)

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Exposure

Healthy

(95%)

Year 1 Year 2 Year 3 thereafter

TB (5%)

Healthy

(92%)

TB (3%)

Healthy

(91%)

TB (1%)

Healthy (approx. 90%)

TB (less than

0.1%/year)

Development of Tuberculosis (the clinician’s view)

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Early bacterial growth arrested at early time point. May (or may not) result in latent infection

Initial exposure

Early bacterial growth not contained. Leads to clinical illness

Subsequent bacterial growth contained. Symptoms abate but latent infection established.

Bacterial growth not contained. Progressive disease unless treated

Reactivation of latent infection at a later point in life

33%

67%

9%

24%

2%

Remain healthy but latently infected

22%

These individuals do not apparently skin-test convert

These individuals generally skin-test convert. They often have characteristic patterns on X-ray.

Response to infection (the immunologist’s view)

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Butajira Hospital

%

%[

N

EW

S

Somali

Oromia

SNNPR

Gambella

Amahara

Afar

Tigray

Addis Ababa

DD

Harare

Benshangul Gumuz

Butajira

Hossana

High TB burden

Study Sites

Hossana Hospital

Study Sites in Ethiopia

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Recruitment in Ethiopia

Index cases (TB, n=76)• smear /culture positive• X-ray confirmed• Symptomatic

Healthy Household Contact (HHC, n=104)• smear/culture negative• X-ray normal• Asymptomatic

Symptomatic Household Contact (XHC, n=58)• smear/culture negative• X-ray often, but not always, suspicious• Symptomatic

Community Control (CC, n=40)• smear/culture negative• X-ray normal• Asymptomatic• No known TB contact

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BloodPBMC Frozen in liquid N2

RNA

Culture with specific antigens

IFN- ELISpot

cDNA

PCR

Plasma

HIVDouble ELISA

Agarose gelOD reading12 bit CCD camera

frozen at -20 0C

IFN- ELISA

ELISA: sTNFR2 IL-10

Ag-specific Ab

Thawed

96 hr Culture supernatant

24/48 hrcultured cells

Real Time PCR

Methods

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TB-specific antigens

M. tuberculosis

Atypical mycobacteria

BCG

M.tuberculosis specific Antigens (100+):

ESAT-6

Common mycobacterial Antigens (1000+)

Ag85A/BRv2031c

Shared TB complex Antigens (4000+)

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Risk of TB in ESAT+ healthy contacts from Ethiopia

35 healthy household contacts of TB patients were tested by ELISA for their response to ESAT-6 or PPD

Over the next two years their clinical status was monitored

Doherty et al., JCM , Feb. 2002

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High ESAT-6 immune reactivity reflects high levels of M. tuberculosis replication

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Time after infection

“positivity” threshold

People who fail to control bacterial replication become ESAT+ and get TB

People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected

People who control initial bacterial replication remain ESAT-, and may or may not be latently infected

“Clinical disease” threshold

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TB HHC CC0

50

100

150

p<0.05

p<0.01

TB HHC CC0

50

100

150 b

p<0.01

p<0.01

TB HHC CC0

50

100

150c

Differential cytokine expression in clinical cohorts

TB HHC CC0

50

100

150

p<0.001

p<0.001

e

TB HHC CC0

50

100

150 b

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Differential cytokine expression in healthy contacts

HHC ESAT- HHC ESAT+ HHC Ag85A- HHC Ag85A+0

1

2A B

p<0.05

10

100

1000

10000

TB HHC CC

ESAT-6 response in clinical groups

+

-

IL-4 mRNA expressed as percentage -actin

HHC ESAT- HHC ESAT+ HHC Ag85A- HHC Ag85A+ 0

75

150 a b

p<0.05

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Differential cytokine expression in community controls


0

0.5

1.0

1.5

IFN- mRNA expressed as percentage -actin


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Change in cytokine expression over time (TB patients)

0.00

0.25

0.50

0.75

1.00Pre-treatment

Post-treatment

IL-4 IFN- -10IL -4IL 2 -12IL

<0.05p

Cytokines in TB patients pre and post treatment

<0.05p

<0.05p

mR

NA

rel

ati

ve

to

-act

in

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Change in cytokine expression over time (contacts)

mR

NA

rel

ati

ve

to

-act

inm

RN

A r

ela

tiv

e to

-a

ctin

IFN- -4IL 2 -4IL -10IL0

50

100

150

<0.05p <0.002p

Cytokines in contacts changing from symptomatic to asymptomatic

IFN- -4IL 2 -4IL -10IL0

50

100

150

Cytokines in contacts changing from asymptomatic to symptomatic

<0.005p <0.005p

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A lowered ratio of IL-4 to IFN- and IL-42 reflects a shift from acute to latent TB

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Th1

?

Th1

Th2

Th1

Th2

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Initial exposure





33%

67%

9%

24%

2%


22%

These individuals do not apparently skin-test convert or become ESAT-6 positive

These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray.

Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN- and IL-12

Immunologically, these individuals tend to express elevated levels of IFN- and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-42 is greatly increased

Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals


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Acute infection Latent infection

Expression of early phase Expression of late phase genesgenes such as Ag85 such as -crystallin and and ESAT-6 the DosR regulon

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Acute Disease

Reactivation of infection

Years after exposure

1-3 4-50

Elimination?

Latent infection

Immune conversion

Latency?

Bacterial response to infection

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10

100

1000

10000

TB HHC LTBI

p<0.001

p<0.001

Rv2031c response in clinical groups

10

100

1000

10000

TB HHC LTBI

ESAT-6 response in clinical groups

IFN- (pg/ml)

Alteration of antigen recognition as disease progresses (ET)

R2 = -0.097

R2 = 0.3688

R2 = -1.0634

0

2000

4000

6000

0 1000 2000 3000 4000 5000ESAT-6 response

RV2031c response

CCHHCTBLinear (CC)Linear (HHC)Linear (TB)

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Alteration of antigen recognition as disease progresses (Ga and NL)

Slo

pe

of

line

ar

reg

ress

ion

no

. o

f sp

ots

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

TB HHC CC0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Clinical status of participants from The Gambia

TB HHC CC0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Clinical status of participants from Ethiopia

Slo

pe

of

line

ar

reg

ress

ion

no

. o

f sp

ots

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

Slo

pe

of

line

ar

reg

ress

ion

IF

N-

fro

m E

SA

T-6

st

imu

latio

n v

s R

v20

31

c

0.0000

0.0005

0.0010

TB TST+Clinical status of participants from the Netherlands

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ratio nHBHA-IFN- /ESAT-6-IFN-

0.00

0.25

0.50

0.75

1.00

100

200

300

400

Active TB Latent TB

Comparison of the IFN- secretions induced by nHBHA and by ESAT-6

Latent TB

Log IFN- conc. (pg/ml)

10

100

1000

10000

100000

nHBHA

Active TB

p< 0.005

p< 0.005

ESAT-6

Active TBLatent TB

Figure kindly provided by Camille Locht and François Mascart

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A lowered ratio of ESAT-6 immune reactivity to Rv2031c reactivity reflects a shift from acute to latent TB

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ES

AT

-6

Rv

20

31

c ES

AT

-6

Rv

20

31

c

ES

AT

-6

Rv

20

31

c

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Initial exposure





33%

67%

9%

24%

2%


22%

These individuals do not apparently skin-test convert or become ESAT-6 positive

These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray.

Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN- and IL-12. They weakly recognise Rv2031c

Immunologically, these individuals tend to express elevated levels of IFN- and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-42 is greatly increased. They strongly recognise Rv2031c

Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals


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Summary

Immunity to M. tuberculosis is dependent on the generation of Th1 immunity, particularly IL-12, IFN- and TNF-

As the bacteria persists in the face of this Th1 response, it begins to alter its proteome towards a pattern characteristic of latency, downregulating some antigens, upregulating others

At the same time, a Th2 response seems to develop

Susceptibility to infection therefore appears to correlate not so much with inability to generate a Th1 response, as with inability to maintain it long term, or perhaps inability to direct it to relevant antigens

We are starting to see evidence that M. tuberculosis-derived antigens are driving some of this Th2 response

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Activation of the immune response (the molecular biologist’s view)

TLR2TLR4

MYD88+MAL

IRAK

TRAF6

NFBNEMO/IKK

TNF-IL-12,IL-18,IL-6, etc

Lipoproteins Peptidoglycans

M.tuberculosis

The activation of the inflammatory cascade is essential for protection.

Defects in this pathway in either humans or animals are associated with extreme susceptibility to mycobacteria

Blocking either IFN- or TNF- after induction of immunity also blocks control of infection

However - M. tuberculosis also needs these cytokines to cause pathology so that it can spread

TIRAP

IRF3IL-18,etc

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PAMP binding

IL-12

IL-12R

IFN- IFN-R

Uptake/Phagocytosis

Lysosome maturation and bacterial killing

Jak/Stat activation

TNF-

TNF-R

Presented

antigen

Specific T cell

proliferation

TCR

T cell Antigen presenting cell

M. tuberculosis

Stat1 activation

IL-18IL-18R

Mycolic acids, lipoproteins

Generation of protective immunity (the cell biologist’s view)

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PAMP binding

IL-12

IL-12R

IFN- IFN-R

Uptake/Phagocytosis


Jak/Stat activation

TNF-

TNF-R

Specific T cell

proliferation

TCR


M. tuberculosis

Stat1 activation

Elevated IL-10

IL-10R

IL-10R

IL-18IL-18R

Mycolic acids, lipoproteins Blocking of

phagocytic maturation

Elevated IL-4/13

Presented

antigen

What can go wrong with protective immunity

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PAMP binding

IL-12

IL-12R

IFN- IFN-R

Uptake/Phagocytosis


Jak/Stat activation

TNF-

TNF-R

Presented

antigen

Specific T cell

proliferation

TCR


M. tuberculosis

Stat1 activation

DC-SIGNIL-10

IL-10R

IL-10R

IL-18IL-18R

PGL Mycolic acids, lipoproteins ESAT-

6/ CFP10

Bacterial lipid-induced IL-4/13

Decoy antigens (27 kDa, PE/PPE family) 19 kDa

LAM

Effect of mycobacterial products on the immune response

Documents

Global burden of tuberculosis